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Effect of matrine on primary human hepatocytes in vitro.

ABSTRACT: Matrine is a bioactive component of the traditional Chinese medical herb Sophora flavescens that has been used in China to treat various kinds of diseases including virus hepatitis. However, the molecular mechanisms underlying its hepatoprotective effects remains elusive. In the present study, primary human hepatocytes were employed to elucidate the protective effects and molecular mechanisms of matrine. We observed that low concentrations of matrine had no significant impact on albumin secretion, but high concentrations (>140 mg/L) of matrine decreased the albumin secretion in hepatocytes. Western blot data indicated that matrine at 140 mg/L at 72 h induced protein expression of CYP2A6, CYP2B6 and CYP3A4. Furthermore, high concentrations of matrine reduced LDH and AST levels and were cytotoxic to hepatocytes, leading to a decreased cell viability and total protein amount. Moreover, low concentrations of matrine, enhanced the ECOD activity and decreased the level of NO2 (-) induced by cytokines in human hepatocytes. Taken together, the present study sheds novel light on the molecular mechanisms of matrine and potential application of matrine in hepatic diseases.

SUBMITTER: Gong X

PROVIDER: S-EPMC4329302 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Effect of matrine on primary human hepatocytes in vitro.

Gong Xiaobing X Gao Yuan Y Guo Guoqing G Vondran Florian W R FW Schwartlander Ruth R Efimova Ekaterina E Pless Gesine G Sauera Igor M IM Neuhaus Peter P

Cytotechnology 20140121 2

Matrine is a bioactive component of the traditional Chinese medical herb Sophora flavescens that has been used in China to treat various kinds of diseases including virus hepatitis. However, the molecular mechanisms underlying its hepatoprotective effects remains elusive. In the present study, primary human hepatocytes were employed to elucidate the protective effects and molecular mechanisms of matrine. We observed that low concentrations of matrine had no significant impact on albumin secretio ...[more]

PMID: 24445680

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Project description:Primary human hepatocytes (PHHs) remain the gold standard for in vitro investigations of xenobiotic metabolism and hepatotoxicity. However, scarcity of liver tissue and novel developments in liver surgery has limited the availability and quality of tissue samples. In particular, warm ischemia shifts the intracellular metabolism from aerobic to anaerobic conditions, which increases glycogenolysis, glucose depletion and energy deficiency. Therefore, the aim of the present study was to investigate whether supplementation with glucose and insulin during PHH isolation could reconstitute intracellular glycogen storage and beneficially affect viability and functionality. Furthermore, the study elucidated whether the susceptibility of the tissue's energy status correlates with body mass index (BMI). PHHs from 12 donors were isolated from human liver tissue obtained from partial liver resections using a two-step EDTA/collagenase perfusion technique. For a direct comparison of the influence of glucose/insulin supplementation, we modified the setup, enabling the parallel isolation of two pieces of one tissue sample with varying perfusate. Independent of the BMI of the patient, the glycogen content in liver tissue was notably low in the majority of samples. Furthermore, supplementation with glucose and insulin had no beneficial effect on the glycogen concentration of isolated PHHs. However, an indirect improvement of the availability of energy was shown by increased viability, plating efficiency and partial cellular activity after supplementation. The plating efficiency showed a striking inverse correlation with increasing lipid content of PHHs. However, 60 h of cultivation time revealed no significant impact on the maintenance of albumin and urea synthesis or xenobiotic metabolism after supplementation. In conclusion, surgical procedures and tissue handling may decrease hepatic energy resources and lead to cell stress and death. Consequently, PHHs with low energy resources die during the isolation process without supplementation of glucose/insulin or early cell culture, while their survival rates are improved with glucose/insulin supplementation.

Project description:AIM:To investigate the effects of electroporation on primary rat hepatocyte and to optimize the electroporation conditions introducing foreign genes into primary hepatocytes. METHODS:A single-pulse procedure was performed at low voltage (220-400 V) but with high capacitance (500-950 microF). Hepatocytes were divided into 4 groups according to the electroporation conditions: group I, 220 V and 500 microF; group II, 220 V and 950 microF; group III, 400 V and 950 microF,and group IV. The control group was freshly isolated hepatocytes and directly cultured under the same conditions as those of electroporation groups. The effects of electroporation on primary rat hepatocytes were detected by trypan blue exclusion (TBE) and MTT analysis. Besides, albumin (Alb), alanine transaminase (ALT) and lactate dehydrogenase (LDH) in the supernatants of cultured hepatocytes were measured by biochemical assay. RESULTS:Between day 1 and day 15 after incubation, primary rat hepatocytes of each electroporation group appeared normal, being the same with those of control group. TBE staining showed that slight hepatocyte damage and high survival rate were found in the electroporation groups and the control group. Cultured for 3, 7, 11 and 15 days, hepatocyte viability was approximately 92.6+/-2.5 %, 89.5+/-3.3 %, 82.0+/-3.5 % and 74.3+/-1.2 %, respectively. MTT analysis indicated that the viabilities of hepatocytes had no significant difference between each electroporation group, and those were similar to that of control group. At the 36th hour after electroporation, Alb, ALT and LDH in the supernatants of control group were 5.3+/-0.1 g x L(-1), 183.7+/-8.4 nkat x L(-1) and 896.8+/-58.5 nkat x L(-1); those of group II were 5.7+/-0.1 g x L(-1), 215.4+/-16.7 nkat x L(-1) and 1063.8+/-51.8 nkat x L(-1); and those of group III were 5.8+/-0.2 g x L(-1), 217.1+/-8.4 nkat x L(-1) and 1063.8+/-10.0 nkat x L(-1). Statistically, the proteins of group II and group III were significantly higher than those of control group (P<0.05), whereas the protein production of group I, Alb, ALT and LDH were 5.3+/-0.2 g x L(-1), 205.4+/-3.3 nkat x L(-1) and 1035.4+/-116.9 nkat x L(-1), were similar to those of control group. At the same time, TBE and MTT analysis indicated that there was no significant cell viability difference between electroporation groups and control group. CONCLUSION:This single-pulse electroporation procedure performed at low voltage (220-400 V) but with high capacitance (950 microF) is one of the optimal choices to introduce foreign genes into primary rat hepatocyte.

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Effect of matrine on primary human hepatocytes in vitro.

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Effect of matrine on primary human hepatocytes in vitro.

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